Pelagic Polychaetes Rosanna Guglielmo1, Alessandro Bergamasco2*, Roberta Minutoli3, Francesco P

Home , Otranto, Poecilochaetidae, Tomopteris

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OPEN The Otranto Channel (South Adriatic Sea), a hot-spot area of plankton biodiversity: pelagic polychaetes Rosanna Guglielmo1, Alessandro Bergamasco2*, Roberta Minutoli3, Francesco P. Patti1, Genuario Belmonte4, Nunziacarla Spanò5, Giacomo Zagami3, Vincenzo Bonanzinga 3, Letterio Guglielmo1 & Antonia Granata3

Composition, density and specimen sizes of pelagic polychaete assemblages were analyzed in the Southern Adriatic Sea. The study was based on fnely stratifed vertical (0–1100 m) and spatial sampling (17 stations) representing spring conditions. Holoplanktonic polychaetes were distributed in both neritic and pelagic waters, although the highest densities were observed along the Otranto Channel. Analysis of the size frequency distribution revealed a trend with depth only for some species. Spatial distribution of holoplanktonic polychaete density was not related to bottom depth, being the organisms mainly concentrated in the epipelagic layer (0–100 m). The most abundant species showed maximum values below or within the thermocline and within the Deep Chlorophyll Maximum or just above it. Relations between polychaete presence and the underlying oceanographic mechanisms regulating the circulation in the Otranto Channel were discussed. The presence of several nondetermined polychaete larvae (e.g. Syllidae) in the pelagic waters at 800–1100 m depths suggests the importance of the role of Levantine waters as main actual and potential carrier of species in the area, though a relevant contribution comes also from North Adriatic dense waters through deep spilling and cascading in the Southern Adriatic pit. These fndings increase the knowledge on holoplanktonic polychaetes ecology within the South Adriatic Sea, and represent signifcant data in the monitoring of changes in biodiversity.

Polychaetes are a large and diversifed group, widely distributed in freshwater and marine habitats, from the intertidal zone to the deepest sediments. Tere are about 9000 nominal polychaete species in the world1, but their diversity is supposed to be much larger and there are many species that have yet to be described. Most of them inhabit the benthic environment, although several species and a few families have evolved to colonize the pelagic realm, showing hyaline body and large appendages for foating and swimming2–4. Holoplanktonic polychaetes are common in marine zooplankton3, although they are not highly important in terms of abundance5. Some species may, at times, be the dominant forms in the plankton community6–8. Tey are widely distributed in the oceans, mainly in the open sea, but they also occur in neritic region3. Pelagic polychaetes inhabit the entire water column, most species are found in the 100 m thick surface layer9, even if several forms have bathypelagic habits10. In spite of their low abundance in the plankton, but in consideration of a wide range of feeding strategies, holoplanktonic polychaetes play an important role in the pelagic food web11,12 and in organic matter minerali- zation13. Teir trophic ecology is complex: (i) most species are active predators and use their quickly eversible proboscis to attack other zooplankters, such as fsh larvae, siphonophores, chaetognaths and appendicularians

1SZN- Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Napoli, Italy. 2CNR-ISMAR (Institute of Marine Sciences, National Research Council), Arsenale - Tesa 104, Castello 2737/F, 30122, Venice, Italy. 3Dipartimento di Scienze Biologiche, Chimiche, Farmaceutiche ed Ambientali, Università di Messina, 98100, Messina, Italy. 4CoNISMA O.U. Lecce, DiSTeBA-University of Salento, 73100, Lecce, Italy. 5Dipartimento di Scienze biomediche, odontoiatriche e delle immagini morfologiche e funzionali, Università di Messina, 98100, Messina, Italy. *email: [email protected]

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(ii) some others are flter-feeders or phytophagous and (iii) only few species are detritivorous13. In turn, they are a basic food, rich in calories, of numerous fshes3 and of predatory large copepods7. Most studies on holoplanktonic polychaetes have emphasized taxonomy, but ecological aspects are poorly known12. Spatial distribution of the families Typhloscolecidae and Tomopteridae in the tropical eastern part of the Pacifc Ocean is available14,15 as a frst step towards understanding the pelagic polychaete fauna in the tropical western Atlantic region16. Many authors have demonstrated a strong correlation between the distribution of planktonic polychaetes and water masses circulation in the South Atlantic and North Pacifc17–19, or suggested that the structure of holoplanktonic polychaete assemblage (oceanic and neritic) could be determined by the feeding habits of the species and their tolerance to the variability in environmental conditions12. As regards Mediterranean Sea, pelagic polychaetes have been largely neglected respect to the benthic polychaete fauna. Monthly changes in composition and abundance of meroplankton and pelagic polychaetes (6 species) of the Cilician Basin shelf waters were reported20, whereas in the checklist of Anellida Polychaeta of the Eastern Mediterranean Sea regions, 14 species of planktonic polychaetes were included from the coasts of Turkey21. More recently, two species of both Typhoscolecidae (Sagitella kowalewski Wagner, 1872 and Typhloscolex muelleri Busch, 1851) and Iospilidae (Phalacrophorus pictus Greef, 1879 and Iospilus phalachroides Viguier, 1886), already included in the checklist of polychaetes of the Italian seas22, have been reported for the frst time in superfcial waters (20–50 m depth) along the northwestern coast of Isola d’Elba (Tuscany)23. Te frst list of all pelagic and benthic polychaetes of the Adriatic Sea was compiled in early nineties24 and included 559 species belonging to 53 families. As for the holoplanktonic polychaetes it is based on several contributions during the last century25–32. Compared with earlier studies in the Southern Adriatic Sea, many papers presented data on participation of polychaetes in total zooplankton9,33–35 focusing the attention on the temporal variation of the abundances of some species of polychaetes in order to hypothesize faunal changes associated with the changes of pelagic waters circulation in the South Adriatic through the Otranto channel and on pelagic polychaete distribution and seasonal dynamics36. More recently, a complete polychaete checklist of the whole Adriatic Sea was compiled37, in which 24 species of holoplanktonic polychaetes were reported. However, information on the vertical distribution of the holoplanktonic polychaetes population in the diferent hydrographically defned sub-basins remains lacking. Tis study aims at describing the composition and abundance patterns of the holoplanktonic polychaete communities in the Southern Adriatic waters, across the Otranto Channel key area and contributes to fll some knowledge gaps thanks to a fnely stratifed spatial samplings up to 1100 m depth. Te general objectives are to shed light on (1) how the pelagic polychaete distribution and size patterns link to environmental conditions and variability such as horizontal gradients and vertical structure of the water column and (2) how the meso-scale oceanographic circulation can modulate the Northern Adriatic and Ionian inputs of species in the Southern Adriatic Basin, through climate-regulated mechanisms. Methods Study area. Te study area includes the Southern Adriatic basin (maximum depth 1200 m), the Otranto Channel (lat 40°N, lon 19°E, 70 km wide) and its interface with the Ionian Sea, a key region for the regulation of the deep overturning cell of the eastern Mediterranean basin38. In particular, the infuence of the Adriatic Deep Water (ADW) outfow through the Otranto Channel triggered by the formation of dense waters in the Adriatic basin during winter intense cooling events has been put in connection with the periodic reversal of the main upper layer circulation in the Ionian Sea and the consequent alternate advection of Atlantic or Levantine waters in the Southern Adriatic basin (the BIoS mechanism)39. Moreover open-ocean deep convection can be responsible during winter for the production of dense water, generating a mixture of the less saline waters from the Adriatic Sea with the more saline and warmer waters originating from the Ionian Sea40. Te role of the Adriatic Sea as the main source of dense water is known to strongly depend upon the atmos- pheric and thermohaline conditions41. In fact, in the early 1990s the Aegean Sea became the main deep-water formation area (the event is known as the Eastern Mediterranean Transient-EMT), with relevant implications for the water mass properties and circulation of the whole levantine basins42,43. Te mechanism seems nowadays re-established and the Adriatic Sea is returning to dominate the eastern deep-water production44. Te surface layer of the Southern Adriatic basin is characterized by the presence of Adriatic Surface Water (ASW), that mainly fows geostrophically along the Italian coast and features a relatively lower salinity due to the freshwater discharges (Po river) and the Ionian Surface Water (ISW), saltier (S > 38.25) and warmer (T > 15 °C) than ASW, that fows into the Adriatic along the eastern side of the Otranto Channel. During winter, the ISW is spread by the dominating south-easternly winds and in spring it occupies almost the whole basin. Trough the same gateway along the eastern border the Levantine Intermediate Waters (LIWs) fow into the Southern Adriatic basin where they undergo local mixing and form the Modifed Levantine Intermediate Waters (MLIWs), characterized by salinity maximum. MLIWs are defned by S > 38.6 and T > 13.5°C and in the Southern Adriatic basin they can be identifed in the layer 150–400 m45. Te impact of LIW infow on the biogeochemical cycles in the Adriatic Sea is substantial, and the fuctuation of a number of physical, chemical, and biological parameters in the Adriatic Sea has been attributed to the LIW ingression9,46,47. In the deepmost layer of the Southern Adriatic basin the Adriatic Deep Water (AdDW) can be found. According to density, two types of AdDW can be distinguished: the North Adriatic Deep Water (NAdDW, σt > 29.2), the newly formed dense water that can reach in 2–3 months directly the Southern basin by fowing along the Italian coast or spilling through the Central Adriatic Jabuka Pit48 and the Southern Adriatic Deep Water (SAdDW, σt > 29.1), considerably warmer and saltier than NAdDW, that forms in the Southern basin by mixing with the overlaying MLIW.

Sampling procedure. Te examined zooplankton samples were collected during the CoCoNet multidisci- plinary oceanographic cruise carried out from 8 to 21 May, 2013 in the Southern Adriatic Sea by the R/V Urania.

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Position Local Time Bottom depth Max sampled depth Station Local date Lat. N Long. E Start End (m) (m) S1 9-May-2013 42°09.994′ 15°39.966′ 20:23 21:37 99 90 L41 10-May-2013 41°59.952′ 16°59.872′ 18:38 20:21 580 550 S3 10-May-2013 42°09.985′ 16°38.059′ 14:05 15:47 178 170 S10 11-May-2013 41°29.780′ 18°22.453′ 23:48 02:07 1123 1096 S7 11-May-2013 42°10.049′ 18°32.310′ 15:28 16:46 190 180 S8 12-May-2013 41°29.971′ 18°50.127′ 04:47 06:35 324 310 S16c 13-May-2013 40°53.072′ 18°57.210′ 11:48 13:09 317 300 S15 13-May-2013 41°02.365′ 18°31.554′ 05:36 07:38 939 900 S22 14-May-2013 40°05.222′ 19°21.708′ 18:03 20:25 965 900 S21 14-May-2013 40°05.008′ 19°08.001′ 22:17 00:28 972 900 S23 15-May-2013 39°40.001′ 19°22.009′ 18:01 20:29 1172 1100 S24 15-May-2013 39°40.004′ 19°08.008′ 22:14 00:23 1089 1000 S20 16-May-2013 40°05.012′ 18°50.071′ 14:50 16:41 738 700 S25 16-May-2013 39°39.917′ 18°22.140′ 04:26 05:59 261 210 S19 17-May-2013 40°26.801′ 18°32.195′ 10:19 11:42 127 100 S14 17-May-2013 41°02.305′ 17°52.030′ 18:02 19:51 699 600 S11 18-May-2013 41°29.991′ 17°34.972′ 07:10 09:25 1137 1060

Table 1. CoCoNet-WP11 May 2013 cruise in the South Adriatic Sea. Stations and sampling dates. Stations and sampling data.

Figure 1. South Adriatic Sea: BIONESS sampling stations during the CoCoNet-WP11 oceanographic cruise, May 8−21, 2013.

Te sampling included 17 stations (Table 1) on the Apulian and Albanian shelves and ofshore waters, including the Strait of Otranto (Fig. 1). Vertical profles of temperature and salinity were collected in each stations with a SBE19 CTD (Sea-Bird Scientifc, USA) to describe the overall oceanographic context of zooplankton samples. A total of 146 zooplankton samples were collected in several layers between the surface and few meters above the seabed, along a 0–1100 m water column. Samples were measured with the electronic multinet EZ-NET BIONESS (Bedford Institute of Oceanography Net Environmental Sampling System)49. Tis 0.25 m2 mouth model was equipped with 10 nets (mesh size 230 μm) and with a multi-parametric probe SBE 911plus (Sea-Bird Scientifc, USA), which continuously recorded temperature, salinity, towing depth and fuorescence. Initial real-time data on depth (m), temperature (°C), salinity and fuorescence (μg L−1 Chl a) were processed with Ocean Data View (ODV) sofware50 to obtain vertical profles. Flow velocity and fltration efciency were monitored by two internal and external fowmeters. Te BIONESS was deployed at low speed along an oblique path to the maximum

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selected depth to be investigated, towed at a speed of 1.5–2 m s−1 until closing, with the simultaneous opening of a new net. Te nets were opened and closed on command from the ship at selected sampling layers according to vertical profles of associated biological and physical parameters. Te fltered volume of water in each layer varied between 15 and 107 m3, according to the thickness of the sampled layer (Table S1). Te number and the thickness of the sampled strata depended on the bottom depth. In the uppermost 100 m depth, the sampled layers were 10–40 m of thickness, and between 100 and 1300 m the layer thickness increased to 50 and 300 m. On board, the samples were preserved in 4% bufered formaldehyde seawater solution.

Laboratory zooplankton analysis. In the laboratory, the pelagic polychaetes were sorted out from the total sample, identifed, and counted under a Leica Wild M10 stereomicroscope. Adult pelagic polychaetes were counted and identifed to the lowest possible taxonomic level following the available descriptions3,51–57. Diferently from all the members of the family Tomopteridae, Alciopidae are mostly very delicate, slender animals, and are rarely found complete; most of them may still be identifed from a head fragment. Terefore, it is in most cases impossible to indicate dimensions and number of segments, but Apstein58 has given a key to the genera by which we may determine a specimen from only one well-preserved parapodium. Tis key forms also the base of Fauvel’s key51 and subsequently other authors55,59,60. Te polychaete larvae have been identifed at family level (Spionidae) or nondetermined. Polychaetes vertical density distribution was estimated from the total specimens counted in any sampled layer divided by the volume of the fltered water and expressed as number of individuals per 100 m3 (ind.100 m-3). In this paper, we present standing crop estimates by a weighted mean of pooled layers between 0 and 200 m, standardized as number of individuals per m2. Randomly selected specimens of abundant complete polychaete species encountered in each sampled stratum were measured for Total Length (TL, from prostomium to pygid- ium) to the nearest millimeter, to obtain minimum–maximum length range and mean length for each species61. Shrinkage due to formaldehyde preservation (ca. 10%) was not considered. A more detailed study was conducted on the vertical distribution of the three species that dominated the polychaetes community. Polychaete abundance and TL patterns across the relevant factors (station, layer depth, habitat type along the column, water mass type) and the main environmental variables (temperature, salinity and chlorophyll) were studied by using R tool packages and the StatGraphics sofware packages. To test for signifcant diferences between and within groups of data, analysis of variance (one-way ANOVA) was employed, coupled with parametric (Fisher’s LSD on means, in case of homogeneity of variances) or non-parametric (Kruskal-Wallis on medians) tests. Te non-parametric Spearman rank correlation test has been used to measure the degree of association between two variables. Moreover, distance-based Redundancy Analysis (dbRDA) was used to describe simi- larity patterns in holoplanktonic polichaetes communities associated with environmental parameters (latitude/ longitude, depth, temperature, salinity and chlorophyll a). Bray-Curtis distance applied to non empty samples was used. All analyses were performed using ‘vegan’ package in R. Sample-based abundance data were used to estimate overall asymptotic richness in the dataset and rarefaction/extrapolation of species richness in the reference samples of the diferent water mass types through the sofware package EstimateS 9.1062. Expected species richness was calculated through the Chao1 estimator (classic formula) and frst order jackknife analysis. Completeness of the samples was evaluated according to the approach proposed by Chao & Jost63. Results Environmental parameters. Te data by the BIONESS were used to defne the spatial variability of environmental parameters (T, S and Chl a) during the spring cruise (CoCoNet 2013) in the Southern Adriatic Sea. Horizontal and vertical variability of thermohaline characteristics in the area evidenced some marked diferences both among stations and along the sampled water column. Te Τ-S profles collected during the campaign by a SBE19 CTD are shown in Fig. 2a. Mid May conditions well represent the middle phase of spring in Southern Adriatic, with a diferent water masses vertical structure among Apulian, ofshore and Albanian stations. In particular, the upper layer up to 100–150 m is dominated by ISW except along the Italian coast, where ASW can be found. Te intermediate layer hosts the MLIW and the deep layer is occupied by ADW originated in the Southern basin (SAdDW) or formed in the Northern basin (NAdDW). According to this structure, the 146 BIONESS samples were labeled to form 4 clusters: 5 samples were assigned to ASW, 71 to ISW, 28 to MLIW and 42 to ADW. Among these latter, 8 were assigned to NADdW and 34 to SAdDW. T-S BIONESS profles provide an overall hydrographical picture of the all investigated area (Fig. 2b), showing an evident stratifed temperature and a marked thermocline that separates the upper layer from the underlaying layers. On average, the Italian side was occupied by colder and less salty waters than Albanian coast. Surface temperature showed increasing trends from Italian (T = 18.90°C, S = 38.85) to Albanian (T = 20.20°C, S = 38.94) side, determined by less salty water masses from Northern Adriatic. A shallow thermocline and a colder temperature were recorded on the Italian side (St. 14, 19; 30–50 m; 15.8–14.5 °C) rather than in the Albanian one (St. 7, 8; 30–65 m; 16.1–15.5 °C). In the pelagic area (St. 15, 16c) the thermocline is deeper (60–90 m) with an average temperature between 15.2 and 14.9°C. In the Otranto Channel (St. 20, 21, 22) the thermocline was recorded between 32 and 65 m, with an average temperature between 16.0 and 15.4°C. In the Ionian waters, at the entrance to the Otranto Channel (St. 23, 24, 25), the thermocline was between 50 and 65 m, and temperature values almost constant (15.4–15.5°C). Chlorophyll fuorescence showed maxima in the layer between 50 m and 80 m in depth for all the stations, except for St. S1 that showed highest chlorophyll a concentration at about 35 m (1.17 mg m−3). Fluorescence profles showed a diferent depth of the Deep Chlorophyll Maximum (DCM). Generally, in the areas close to the coast and in Otranto Channel this maximum was detected at 60 m, but with very diferent max chlorophyll a values: 0.696 mg m−3, 1.54 mg m−3 and 1.12 mg m−3, in the Apulian, Albanian side and Otranto Channel, respectively. In

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Figure 2. Oceanographic context during the CoCoNet Cruise in Southern Adriatic Sea (May 2013). (a) θ-S diagram of the collected CTD profles. Colors indicate the depth (m). Water mass types are highlighted. (b) Vertical profles of temperature (°C), salinity and chlorophyll a (µg/L) from all the BIONESS sampled stations. Vertical scale is expanded in the 0−200 m layer (where original data are pooled every 10 m) with respect the remaining of the water column (where original data are pooled every 100 m).

the ofshore waters the DCM was slightly deeper (66 m, 0.72 mg m−3), while in the Ionian stations it was found at about 73 m (0.95 mg m−3). Below 120 m in depth, the chlorophyll a concentration dropped to very low values in all of the stations.

Holoplanktonic polychaetes. Density and species composition. Mean total zooplankton density, among the 17 sampled stations, was 44,000 ind.100 m−3 (CV = 58%). Copepods were the most abundant taxon representing from the 72 to 96% of the total zooplankton, with a mean density of 40,000 ind.100 m−3 (CV = 59%), then Chaetognatha (1400 ind.100 m−3; CV = 113%), Ostracoda (617 ind.100 m−3; CV = 84%), Siphonophora

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Density Percent Size range X Species (ind./100 m3) % N (mm) (mm) (±SD) n Holoplanktonic Lopadorrhynchidae Claparède, 1870 Lopadorrhynchus brevis Grube, 1855 0.13 0.20 9 1.29–2.23 2.00 0.47 3 Lopadorrhynchus uncinatus Fauvel, 1915 0.13 0.20 9 2.11–16.47 10.37 7.48 4 Maupasia coeca Viguier, 1886 0.83 1.25 58 1.76–3.76 2.50 0.68 14 Pedinosoma curtum Reibisch, 1895 0.04 0.06 3 4.00 4.00 — 1 Pelagobia longicirrata Greef, 1879 0.13 0.20 9 1.41–2.94 2.35 0.64 4 Alciopidae Ehlers, 1864 Alciopina parasitica Claparède & Panceri, 1867 0.04 0.06 3 4.35–6.47 5.76 1.22 2 Krohnia lepidota (Krohn, 1845) 8.83 13.36 623 1.17–7.64 3.07 1.18 115 Naiades cantrainii Delle Chiaje, 1830 1.74 2.63 123 2.94–13.52 5.33 2.94 31 Plotohelmis capitata (Greef, 1876) 0.07 0.10 5 6.11 6.11 — 1 Plotohelmis tenuis (Apstein, 1900) 0.05 0.07 3 3.17–7.64 6.15 2.58 2 Rhynchonereella gracilis Costa, 1864 0.37 0.56 26 1.18–5.64 4.14 1.29 6 Rhynchonereella moebii (Apstein, 1893) 0.05 0.07 3 9.29 9.29 — 1 Vanadis crystallina Greef, 1876 2.58 3.91 182 1.17–20.58 6.57 5.16 41 Vanadis formosa Claparède, 1870 1.99 3.01 140 2.92–20.00 6.64 3.76 44 Tomopteridae Johnston, 1865 Enapteris euchaeta Chun, 1888 0.05 0.07 3 12.35 12.35 — 1 Tomopteris apsteini (Rosa, 1908) 0.16 0.24 11 2.35–23.52 11.09 9.75 6 Tomopteris catharina (Gosse, 1853) 0.16 0.24 11 2.35–11.76 8.27 3.08 9 Tomopteris ligulata Rosa, 1908 19.41 29.38 1370 1.17–7.41 2.90 0.95 383 Tomopteris pacifca (Izuka, 1914) 7.28 11.03 514 1.17–7.05 3.67 1.12 154 Tomopteris planktonis Apstein, 1900 2.53 3.83 179 2.11–6.00 3.85 0.98 32 *Tomopteris sp. 14.79 22.38 1044 0.70–5.29 3.06 0.97 203 Typhloscolecidae Uljanin, 1878 Sagitella kowalevskii Wagner, 1872 1.83 2.78 129 2.35–7.17 5.32 1.25 42 Travisiopsis lanceolata Southern, 1910 0.35 0.53 25 2.35–4.94 4.00 0.86 7 *Travisiopsis sp. 0.26 0.40 18 3.76–4.11 3.99 0.20 2 Meroplanktonic Poecilochaetidae Hannerz, 1956 Poecilochaetus serpens larvae Allen, 1904 0.03 0.04 2 3.17 3.17 — 1 Syllidae Grube, 1850 Syllidae larvae 1.18 1.78 83 2.11–3.29 2.63 0.40 13 Nondetermined polychaete larvae 1.07 1.62 76 1.76–2.58 2.17 0.30 32

Table 2. Holoplanktonic and meroplanktonic polychaete species identifed in the study area: total mean weighted density over all stations, per cent contribution, number of counted specimens (N), size range with mean value (X), standard deviation (±SD) and number of specimens measured (n). *Remarks: Juvenile specimens not assigned certainly to a species.

(490 ind.100 m−3; CV = 121%) and Amphipoda (300 ind.100 m−3; CV = 115%) were found in decreasing order. Pelagic polychaetes represented only the 0.38% of the total zooplankton community and exhibited a mean density of 66 ind.100 m−3 in 102 non-empty samples (out of 146 in total). Overall 1154 specimens were diagnosed at species level. Regarding composition, 22 species belonging to 4 families (Tomopteridae, Alciopidae, Typhloscolecidae, Lopadorrhynchidae) were identified (Table 2). Tomopteridae was the most abundant family with 44.4 ind.100 m−3 representing about two-thirds of total polychaete density. Among the 6 species belonging to the genus Tomopteris, T. ligulata Rosa, 1908 was the most abundant (19.4 ind.100 m−3) one and the most frequently observed (35% occurrence in the non-empty samples), followed by T. pacifca (Izuka, 1914) (7.3 ind.100 m−3) and T. planktonis Apstein, 1900 (2.5 ind.100 m−3). Only rare specimens of Tomopteris apsteini (Rosa, 1908), Enapteris euchaeta Chun, 1888 and T. catharina (Gosse, 1853) were observed whereas juvenile specimens of this genus were difusely present throughout the study area and reached the maximum density of 14.8 ind.100 m−3 (as Tomopteris sp.). Alciopidae ranked second in density with about 15.7 ind.100 m−3 (23.8% of the polychaete community). Among the 9 species identifed within this family the most abundant was Krohnia lepidota (Krohn, 1845) (8.8 ind.100 m−3) occurring in 28% of the non-empty samples, followed by Vanadis crystallina Greef, 1876 (2.6 ind.100 m−3), Vanadis formosa Claparède, 1870 (2.0 ind.100 m−3) and Naiades contrainii Delle Chiaje, 1830 (1.7 ind.100 m−3). Few specimens of the other fve species (Alciopina parasitica Claparède & Panceri, 1867, Plotohelmis capitata (Greef, 1876), Plotohelmis tenuis (Apstein, 1900), Rhynchonereella gracilis Costa, 1864 and Rhynchonereella moebii (Apstein, 1893)) together constituted only 0.20%.

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Typhloscolecidae was the third family in density with 2.4 ind.100 m−3 (3.7% of the polychaete community). Among them Sagitella kowalewski was the most abundant species (1.8 ind.100 m−3), while Travisiopsis lanceolata Southern, 1910 and Travisiopsis sp. appeared with only few specimens. Lopadorrhynchidae family, with 1.3 ind.100 m−3 (1.9% of the polychaete community), was represented by 5 species with very few specimens among which Maupasia coeca Viguier, 1886 resulted the most abundant and frequent one. Among these species, fve were recorded in the South Adriatic for the frst time: Alciopina parasitica, Pedinosoma curtum Reibisch, 1895, Plotohelmis capitata, Plotohelmis tenuis, Rhynchonereella moebii (Table 3).

Horizontal and vertical distribution. Holoplanktonic polychaetes were distributed in the entire study area (Fig. 3a). Most of the species were found in the 0–200 m stratum, so we considered this layer to calculate the standing crop of the entire polychaete community in the whole study area. Higher densities were recorded in the stations located in Otranto Channel, with the highest value in the Station 22 (2902 ind. m−2). Relatively few specimens were found along the Italian coast and in the ofshore waters, while the stations along the Albanian shelf have shown higher values (Fig. 3b). Tis West-to-East gradient is statistically signifcant (ANOVA Abundances by Longitude, p < 0.001, Kruskal-Wallis) and holds even if the analysis is restricted to the 0–200 m layer, suggesting that the main part of the community occupies the upper part of the water column: in fact, density decreases with increasing depth (Spearman’s rank correlation r = −0.21, p < 0.012, N = 146). Te horizontal distribution of the most abundant species in the 0–200 m layer is shown in Fig. 4. T. ligulata showed a wide distribution (positive records in 10 stations), with highest densities along the Albanian shelf (St. S7, 408 ind. m−2). Tomopteris sp. (recorded in 12 stations) has shown maximum values in the Station S8 (338.7 ind. m−2), while K. lepidota (11 stations) exhibited the highest density in the Station S22 (347.3 ind. m−2), T. pacifca (7 stations) in Sts. S20 and S24 (124.6 and 105 ind. m−2 respectively), V. crystallina (9 stations) in Sts. S7 and S23 (39.1 and 34.8 ind. m−2, respectively). T. planktonis was collected only in 3 stations, showing the highest values in the Stations S21 and S23 (71.6 and 75.9 ind. m−2, respectively). Vertical range distribution, depth of occurrence and density peak of polychaete species for all sampled stations were shown in Fig. 5. Five species (plus Syllidae larvae) occupy the 0–100 m layer, four species the 0–200 m layer (plus nondetermined polychaete larvae), four species the 0–400 m layer, six species 0–600 m layer, 1 species 0–800 m, 4 species 100–200 m and only Pedinosoma curtum the layer 400–600 m. Te maximum depth range (0–1100 m) was recorded by undetermined polychaete larvae. Although with diferent vertical distribution ranges, all species show the maximum density in the epipelagic layer within 200 m, of which about 80% in the 0–100 m layer and only 5 species between 100 and 200 m depth.

Relations with the environmental parameters and water mass structure. As expected the polychaete presence was linked to the water mass structure, with a signifcantly higher density within the samples assigned to ISW respect to ASW, MLIW and ADW (Kruskal-Wallis on medians, p < 0.005) while the comparisons among layers (Surface, DCM, Intermediate, Deep) didn’t highlight signifcant diferences. Figure 6 shows the constrained classifcation produced by dbRDA analysis. Te frst two axes (both signifcant, p < 0.001, ANOVA by axis) explain 87% of the ftted model. Environmental covariates signifcantly infuence the holoplanktonic polichaetes assemblage (Longitude and Depth, p < 0.001; Chlorophyll a, p < 0.01, ANOVA by term). Te frst quarter is associated to the increasing longitude, i.e. towards the southern stations along the Albanian coast, where ISW and MLIW dominate the water column; the species mainly correlated with dbRDA ordination and covariates are in this quarter Krohnia lepidota (that dominate in St. S22), Tomopteris sp. and T. planktonis. Te lef half plane is mainly associated to an increase of depth (second quarter) and a decrease of longitude (third quarter), corresponding to an increase of latitude, due to the shape of the Adriatic Sea. Te stations, mainly the pelagic ones and those along the Italian coast, are characterized by the predominant presence of ADW and MLIW. No species belonging to the three most abundant genera were mapped in this portion of the plane. Finally, the fourth quarter is associated to relatively lower depth and an increase of chlorophyll a. Te stations mapped here mainly belong to the Surface and DCM layers of the ISW along the northern Albanian coast, that are characterized by the presence of Tomopteris ligulata and T. pacifca, and to few MLIW. Considering all the sampled stations, polychaete density was positively correlated to temperature and salinity, with signifcant Spearman’s coefcients (r = 0.37, p < 0.0001 and r = 0.43, p < 0.0001 respectively) whereas a weak correlation was found with chlorophyll a concentration (r = 0.11, p = 0.2). Considering the relation between environmental parameters and the six most abundant species separately, only T. planktonis abundance was not afected by temperature and salinity, and Tomopteris sp. by salinity. Te interrelationships between the distribution of the three most abundant species (T. ligulata, K. lepidota, T. pacifca) and the environmental parameters (temperature, salinity and fuorescence profles) in the Otranto Channel transect (Sts. S20, S21, S22) are presented in Fig. 7. Te three species showed the maximum density below (K. lepidota) or within the thermocline (T. ligulata and T. pacifca) and within the DCM (K. lepidota) or just above the DCM (T. ligulata and T. pacifca). Tis last species was not recorded in St. S21. Looking at the day-night vertical distribution of K. lepidota, in St. S20 and S22 (diurnal) there is a single subsurface maximum in DCM correspondence, while in St. S21 (nocturnal) the population is divided into two parts (a superfcial max and a sub-DCM part).

Size distribution of polychaete assemblage. Te size distribution of the measured total length of the specimens is shown in Fig. 8a. About 75% of the specimens was in the range from 2.0 to 4.5 mm, whereas only 0.5% of the specimens has a total body length beyond the 15.0 mm. Te overall shape of the curve indicates a certain degree of bimodality, in particular for the families Alciopidae and Typhloscolecidae, with the frst maximum around

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Southern Adriatic Sea Tis Mikać Batistić et al. Species Study (2015) (2004, 2007, 2012) Synonymised names Lopadorrhynchidae Claparède, 1870 Lopadorrhynchus brevis Grube, 1855 X X Lopadorrhynchus uncinatus Fauvel, 1915 X X Maupasia coeca Viguier, 1886 X X Pedinosoma curtum Reibisch, 1895 X Pelagobia longicirrata Greef, 1879 X X X Pontodoridae Bergström, 1914 Pontodora pelagica Greef, 1879 X Iospilidae Bergström, 1914 Phalacrophorus pictus Greef, 1879 X Alciopidae Ehlers, 1864 Alciopina parasitica Claparède & Panceri, 1867 X Krohnia lepidota (Krohn, 1845) X X X =Callizonella lepidota Naiades cantrainii Delle Chiaje, 1830 X X Plotohelmis capitata (Greef, 1876) X Plotohelmis tenuis (Apstein, 1900) X Rhynchonereella gracilis Costa, 1864 X X =Callizona nasuta Rhynchonereella moebii (Apstein, 1893) X Torrea candida (Delle Chiaje 1828) X Vanadis crystallina Greef, 1876 X X X Vanadis formosa Claparède, 1870 X X X Tomopteridae Johnston, 1865 Enapteris euchaeta Chun, 1888 X X =Tomopteris euchaeta Tomopteris apsteini (Rosa, 1908) X X =T. scolopendra Tomopteris catharina (Gosse, 1853) X X X =T. helgolandica Tomopteris cavallii Rosa, 1908 X Tomopteris ligulata Rosa, 1908 X X Tomopteris pacifca (Izuka, 1914) X X X =T. elegans Tomopteris planktonis Apstein, 1900 X X Typhloscolecidae Uljanin, 1878 Sagitella kowalevskii Wagner, 1872 X X X Travisiopsis lanceolata Southern, 1910 X X Typhloscolex muelleri Busch, 1851 X X Number of species 22 17 13

Table 3. List of holoplanktonic polychaete species reported for the Southern Adriatic Sea by this study and available literature.

2.5–3.0 mm, that can be assigned to juveniles or larval forms (e.g. Syllidae and undetermined taxa) and the second one around 5.5–6.0 mm, possibly related to the adults. Regarding the individual total lengths (Table 2), Tomopteris apsteini showed the largest size range (2.35– 23.52 mm) and also the longest specimen (23.52 mm). Te smallest individuals belonged to the undefned species of Tomopteris genus (0.7 mm), instead the smallest size range (3.76–4.11 mm) belonged to undefned species of Travisiopsis genus. Te largest mean size (12.35 mm) belonged to Enapteris euchaeta and the smallest (2 mm) to Lopadorrhynchus brevis Grube, 1855. Specimen size was found to be dependent on the capture depth (Fig. 8b). Overall medians of body lengths were found to be signifcantly diferent among the 8 tested layers (ANOVA Kruskal-Wallis, p < 10–6). On average, body lengths were relatively higher in the DCM layer (4.17 ± 2.01 mm) than the Surface one (Fisher LSD test, 99% Bonferroni interval) and maxima in the layer 300 to 600 (> 5.1 mm). At surface the lower lengths are due to the predominance of Tomopteridae (Tomopteris juveniles) against Alciopidae (Khronia and Vanadis), that prefer the underlying layer, and to the presence of several larval forms (e.g. Syllidae) in the ISW that largely occupy the surface waters (Fig. 9a,b).

Overall and water-mass-related species richness. Te sampling efort in the seventeen stations allowed the collection of 146 samples. About 30% of them mainly referring to the Italian shelf and outer shelf did not contain pelagic polychaete specimens at all suggesting a patchy or zonal distribution of this taxonomic group correlated to the oceanographic settings and the circulation patterns. Te reference overall sample (sized n = 1154 identifed specimens, including 6 singletons and 2 doubletons) exhibited a relevant degree of completeness (99%) well

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Figure 3. Spatial distribution of holoplanktonic polychaetes in the study area. (a) Total polychaete density (ind. m−2) integrated in the 0−200 m layer. (b) Polychaete density in each sample (ind. 100 m−3) assigned to the diferent water mass types present in the stations.

representing the Southern Adriatic pelagic polychaete community in spring. Te expected species richness can be estimated to be 25% in excess with respect to the observed one (36 ± 10 species, Chao1 estimator, classic formula). First order jackknife analysis gave an estimated richness of 36 ± 3 species too. To correctly compare species richness in uneven groups of samples64,65, rarefaction/extrapolation curves based on the reference samples in each water mass were plotted (Fig. 10). ISWs exhibit the greatest extrapolated richness, signifcantly higher than MLIWs (p < 0.05) with which there is no overlap between the 95% confdence intervals. Intermediate value can be assigned to ADW and the lowest to ASW, probably depending also on the extremely uneven sampling in these latter waters. Te most relevant diference is that while the extrapolated trend of ISW richness does not show any plateau at a size near the reference sample and far beyond, ADW and MLIW appear to express their asymptotic richness already at 300–400 individuals (17 and 10 species, respectively). Discussion To our knowledge, informations on pelagic polychaete community composition in the South Adriatic Sea are very scarce since available zooplankton data mainly refer to copepods or zooplankton communities9,33,34,66. Due to the scarce numerical importance, their species composition is likewise sometimes disregarded in quali-quantitative zooplankton analyses or reported with basic taxonomic detail67. Tirteen pelagic polychaete species were reported by Batistić et al.9,33,34 whereas seventeen species were more recently included in a review dated 201537 (Table 3). Probably as a consequence of the sampling efort (146 samples in a wide geographic area), a higher biodiversity of

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Figure 4. Density (ind. m−2) in the 0−200 m layer of the most abundant polychaete species.

pelagic polychaetes was found in this study: fve species were recorded for the frst time in this area out of a total of twenty-two species, in addition to Tomopteris sp., Travisiopsis sp., Syllidae larval specimens and nondetermined polychaete larvae, with numerical dominance of T. ligulata, Tomopteris sp., K. lepidota, T. pacifca, V. crystallina and T. planktonis. Hence, to date the overall observed species richness of holopelagic polychaetes within the South Adriatic Sea accounts for twenty-seven species. Interestingly, some species among the ones already recorded34 and suggested as possible indicators of the hydroclimatic changes in Southern Adriatic68, were not found in this study: Phalacrophorus pictus, that is considered a cold species18, more commonly found in neritic (2–10 m layer)

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Figure 5. Depth of occurrence and of maximum density for all pelagic polychaete species recorded in the sampling stations.

Longitude Kro.lep

Tom.sp

) Depth

Tom.pla

Sag.kov

Van.for 01 024 Van.cri Tom.pac

Chlahla Tom.lig -2 db-RDA Axis 2 (34% of fitted variation

ADW ASW ISW -1

-4 MLIW

-4 -2 024

db-RDA Axis 1 (46% of fitted variation)

Figure 6. Constrained classifcations by distance-based Redundancy Analysis (db-RDA) ordination diagram of the holoplanktonic polychaete species in Southern Adriatic Sea. Blue arrows indicate signifcant explanatory variables (longitude, depth and chlorophyll a). Te samples are labeled according to their water-mass cluster membership. Te 8 species belonging to the three most abundant genera (90%) are shown: Tomopteridae (red), Alciopidae (green), Typhloscolecidae (blue). Tom.lig: Tomopteris ligulata; Tom.sp: Tomopteris sp.; Tom.pla: Tomopteris planktonis; Tom.pac: Tomopteris pacifca; Kro.lep: Krohnia lepidota; Van.for: Vanadis formosa; Van. cri: Vanadis crystallina; Sag.kov.: Sagitella kovalewskii.

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Figure 7. Vertical distribution (ind. 100 m−3) of the three most abundant polychaete species and profles of temperature, salinity and fuorescence collected by BIONESS at the Otranto Channel transect. Note the diferences in the density scale. Stations S20 and S22 were sampled during daytime, station S21 was sampled at night.

than pelagic waters69, and Pontodora pelagica Greef, 1879 that, conversely, is considered a warm species70 and classifed as “rare” during winter samplings69,71. Regarding specimen size range, analysis of the size frequency distribution demonstrated a trend in depth distribution by size only for some species. Considering the three most abundant species, T. ligulata, K. lepidota and T. pacifca, only the frst one increased in size with the depth. It should be possible that the older specimens tend to be distributed in deeper layers, instead the smaller specimens inhabit the more superfcial layers for

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Alciopidae Lopadorrhynchidae Syllidae Typhloscolecidae Family Larvae undet. Poecilochaetidae Tomopteridae

150 s

100 Number of specimen

0 a) 0510 15 20 Total Length (mm)

0-40

40-80

80-150 ) 150-200

200-300 Depth Range (m

300-400

400-600

600-800 b) 0510 15 20 Total Length (mm)

Figure 8. Pelagic polychaetes community collected in Southern Adriatic Sea. (a) Size distribution of measured Total Length of the specimens (N = 1154) and (b) its dependence on the capture depth range (for each layer, boxplot and medians are indicated; dots represent the single specimens).

trophic reasons12. Some hypotheses can be made on the presence of nondetermined larvae found in the deeper pelagic waters. First of all, wind-induced intense cooling at the surface has been already indicated to trigger short-duration open ocean convection events able to locally afect the vertical distribution of zooplankton in Southern Adriatic34. Furthermore, larval stages dominate the marine-snow-associated community, with polychaete larvae being one of the most important players in term of biomass72. Polychaete larvae can take advantage of the buoyancy of marine snow for their dispersal and utilize marine snow as a transport vehicle and as a food source. Tis behaviour could justify the abundance of nondetermined larvae found in this study in the pelagic water at 800–1100 m depths. Te comparison of the species richness in the diferent water masses suggests that ISWs are the main actual and potential carrier of species in the area, though a relevant contribution comes also from ADW that could feed through deep spilling and cascading the deeper basins of the Southern Adriatic with larval or juvenile forms present in the Northern Adriatic, so providing transient windows of remote connectivity near the seabed, espe- cially in late winter-early spring period. Similarly to ISW, the overall species accumulation curve did not reach an asymptote neither within the size of the reference sample nor in the extrapolation to a 5-time size (i.e. 5000 individuals), suggesting a remarkably greater species richness for pelagic polychates in the whole area, in the order of at least 50 species. During the sampling period, holoplanktonic polychaetes were distributed in both neritic and pelagic waters, although total polychaete density was highest along the Otranto Channel. Species densities were not related to

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Alciopidae Lopadorrhynchidae Syllidae Typhloscolecidae Family Larvae undet. Poecilochaetidae Tomopteridae

10 Total Length (mm)

0 a) ADW ASW ISWMLIW Water Mass Type

10 Total Length (mm)

0 b) Surface DCM Intermediate Deep Layer

Figure 9. Pelagic polychaetes of the Southern Adriatic Sea. Total Length of the specimens gouped by families versus (a) the Water Mass Type and (b) the Layer. Boxplot, medians and outliers are indicated.

station bottom depth. In fact, the spatial distribution pattern did not change considering only the density data from 0–200 m layer. Te number of collected species was lower in the stations on the Italian side respect the Albanian one. Tis fact has been underlined as a general trend also on microzooplankton73, neuston67 and fsh larvae74 and could be the consequence of a lower injection of nutrients and organic material on the Italian side, where rivers are typically absent46. In addition to the highest total density, the Otranto Channel waters exhibited also the highest number of species (S20: 16 species; S23: 13 species; S21: 11 species), likely related to the hydro- logical functioning of this area. A relation between polychaete spatial density and Otranto Channel water circulation can be hypothesized. Due to its morphology the Strait plays in fact a key role in controlling the exchange of water masses and related properties between adjacent basins75. Mantziafou & Lascaratos reported the maximum water volume transport in spring and minimum in autumn, with a consequent strong hydrodynamism along the Otranto Channel76. Our study was carried out in May, therefore the stations in correspondence of the Otranto Channel were possibly interested by relevant hydrodynamic exchanges that promoted the dragging and accumulation of higher concentration of polychaetes. It was reported that the geographical location of holoplanktonic polychaetes can be correlated to productivity and main water masses14,77,78, showing assemblages in areas of high primary and secondary production66,79–84. All the species found in the present study (except Pedinosoma curtum that represents only the 0.06% of polychaete community) were more abundant in the upper 200 m, and the main part (nineteen of twenty-two species) in the upper 100 m. As already reported, holoplanktonic polychaete species are distributed in the epipelagic region of the water column5,8,9,71,85,86, most probably because this layer is characterized by a large supply of food, with highest concentration of phytoplankton, microzooplankton and

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15 Species Richness

0 0 100 200 300 400 500 600 700 800 900 1000 Number of individuals

ISW ADW MLIW ASW

Figure 10. Rarefaction/extrapolation curves of species richness as functions of the number of individuals in the four water types (ISW: Ionian Surface Water; ADW: Adriatic Deep Water; MLIW: Mixed Levantine Intermediate Water; ASW: Adriatic Surface Water). Reference samples are indicated by solid circles, rarefaction by solid lines, extrapolation by dashed lines. Shaded areas indicate the 95% confdence intervals around each curve.

copepods87,88. Surely, the most important gas in water is oxygen, as its role in metabolic processes is essential to all forms of life and it afects the distribution of pelagic organisms at several spatial scales12. In the present study, the presence of a maximum of chlorophyll a concentration at about 50–80 m allowed us to identify at this layer both a maximum of food availability (phytoplankton) and of oxygen concentration, thus justifying the density of zooplankton and of carnivorous polychaeta in particular. Furthermore pelagic polychaetes were found to be distributed mostly at the thermocline89, as observed in this study for the three most abundant species. Relations between polychaete density and environmental features were investigated. Polychaete distribution patterns were positively correlated with temperature and salinity. Total polychaete density was higher at stations with higher temperature and salinity values, in correspondence of Otranto Channel. Te intensity of Eastern Mediterranean infuence into the Adriatic depends on the advection of Levantine Intermediate Water, which is controlled by the pressure distribution over the wider area; when the infow into the Adriatic is strong, one indicator is just the higher salinity33,90. High densities of pelagic polychaete were already observed in upwelling regions with high salinity91. Vertical polychaete distribution followed the same trend, concentrating in warmer waters in correspondence of or above the thermocline. Pelagic polychaetes distributed mostly at the thermocline (30–100 m), and at the upper and lower Oxygen Minimum Zone (OMZ) interface89,92. No relation was found with chlorophyll a, surely because the six most abundant species, representing all together more than 80% of the community, belonged to Tomopteridae and Alciopidae families thus showing carnivorous habits93 and possibly approaching the phytoplankton-rich layer only in few hours of the day (during vertical migrations). Tomopteridae distribution is worldwide, including oceanic and near-shore waters, from the surface to a few hun- dred meters depth1,12. Feeding habits of Tomopteridae are variable. Some species show a short pharynx and ingest the whole prey or suck out the body fuids, whereas other species miss prey-catching organs and eat microscopic preys3,12. Conversely Alciopidae are long, slender and active free-living predators. Compared with earlier data9,34,35, in this study there was a marked change in polychaete dominant species assemblages collected in the open South Adriatic. Te species that were abundant in this research (Tomopteris ligulata, Krohnia lepidota and Tomopteris pacifca (= T. elegans)) were absent or poorly represented earlier in spring season (April and May). In particular, Pelagobia longicirrata dominated in the whole water colum in April 19939, while no specimens were found in May 199535. Te very few specimens of P. longicirrata (0.20%) collected in this campaign could confrm the results of Batistić35 according to which this species must be considered a cold species. Experimental observations show that the Northern Ionian Gyre (NIG) reverses on multiannual scale39. During its anticyclonic phase (e.g. 2006–2010), the Atlantic Water meanders in the northernmost part of the Ionian Sea and induces a general decrease in salinity in the Southern Adriatic94. In 2011 the NIG became cyclonic95 so favouring the advection of saltier and warmer levantine water in the Southern Adriatic. Afer a rapid and short inversion in 2012 due to the harsh winter, the NIG circulation returned again to be cyclonic at the beginning of 201395. Tis scenario is compliant with the dominance of ISW in the Southern Adriatic as observed during the present study and the very low presence of P. longicirrata in our samples. Terefore, in our study the entry of warmer and saltier water favored warm species such as T. elegans and non P. longicirrata that presents a signifcant negative correlation with temperature35. In conclusion, we agree with Batistić35 that these faunal changes can be associated with the change of NIG circulation and, consequently with the periodic modulation of the infow of Atlantic Water (MAW) or Levantine Water into the Southern Adriatic.

Received: 26 September 2019; Accepted: 29 November 2019; Published: xx xx xxxx

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